SIGNALS FOR COMMITMENT TO RADIATION INDUCED APOPTOSIS

电离辐射(IR)和所有常用于癌症治疗的化疗药物被认为最终会通过引发一种称为细胞凋亡的细胞死亡来杀死肿瘤细胞。因此，控制细胞凋亡的基因调控的改变可能在决定肿瘤的相对放射抗性方面发挥重要作用。这项研究的目的是调查导致IR诱导细胞死亡的关键分子信号。BCL-2/CED-9家族蛋白、ICE/CED-3家族蛋白(Caspase)和CED4/Apa-F1蛋白是细胞凋亡的基本调节因子。然而，这些蛋白质相互作用调节哺乳动物细胞死亡的确切机制尚不清楚。我们的初步研究表明，IR诱导的细胞凋亡与多个半胱氨酸天冬氨酸酶的激活有关，这些半胱氨酸天冬氨酸酶可以蛋白水解性地裂解细胞蛋白，包括Bcl-2。矛盾的是，异位表达的Bcl-2阻止了caspase的激活和自身的切割。本提案将阐述Bcl2的双重功能：i)作为与caspase和APAF-1相互作用的对接蛋白，或ii)作为线粒体相关蛋白，介导该细胞器中的凋亡变化。作为模型系统，我们将使用具有差示辐射敏感性和caspase激活动力学的多发性骨髓瘤细胞系。目前的建议有三个特定的目标：特定的目标1：研究caspase 3切割Bcl2的机制：这个目标将检查：(I)bcl2突变和切割改变的影响，(Ii)这种切割的一般性，和(Iii)Bcl2蛋白水解性切割的机制。特异性目的2：确定Bcl2与细胞凋亡的关键蛋白之间的相互作用。这些相互作用将在没有洗涤剂的情况下进行检查，在这种条件下，蛋白质结构将被保留，使用体内的Bcl-2裂解。蛋白质之间的直接相互作用将通过免疫定位和免疫沉淀结合免疫印迹分析来检验。我们还将在被设计为诱导表达Bcl2的细胞以及将Bcl2定位于特定亚细胞位置的细胞中检测Bcl2的相互作用。特定目的3：为确定细胞凋亡所必需的线粒体事件的调控作用，这些实验将评估：(I)细胞色素c的释放和胞浆复合体的激活，以及(Ii)线粒体膜电位和线粒体通透性孔道转变在IR和Bcl2信号传递中的作用。我们的长期目标是确定激活caspase导致细胞凋亡所必需的关键细胞信号。通过了解IR触发的信号和细胞参与细胞凋亡的临界点，我们最终可能能够特异性地增强caspase的激活和细胞死亡，以克服放射治疗中的辐射抵抗。

Ionizing radiation (IR), and all chemotherapeutic drugs commonly used in the treatment of cancer, are thought to ultimately kill tumor cells by triggering a form of cell death called apoptosis. Consequently, altered regulation of the genes that control apoptosis may play an important role in determining the relative radioresistance of tumors. The objective of this research is to investigate the critical molecular signals responsible for the commitment to IR-induced cell death. Bcl-2/CED-9 family proteins, ICE/CED-3 family proteases (caspases), and the CED4/Apa-f1 protein represent the basic regulators of apoptosis. However, the precise mechanism by which these proteins interact to regulate cell death in mammalian cells is unclear. Our preliminary studies indicate that IR-induced apoptosis is associated with activation of multiple caspases, which proteolytically cleave cellular proteins, including Bcl-2. Paradoxically, ectopic expression of Bcl-2 prevents caspase activation and its own cleavage. The present proposal will address the dual functions of Bcl-2: i) as a docking protein interacting with caspases and Apaf-1, or ii) as a mitochondrial associated protein that mediates apoptotic changes in this organelle. As a model system we will use multiple myeloma cell lines with both differential radiation sensitivity and kinetics of caspase activation. There are three specific aims of the current proposal: SPECIFIC AIM 1: To study the mechanism of Bcl-2 cleavage by caspase 3: This aim will examine: (i) the effect of bcl-2 mutations with altered cleavage, (ii) the generality of this cleavage, and (iii) the mechanism of Bcl-2 proteolytic cleavage. SPECIFIC AIM 2: To determine the interactions between Bcl-2 and proteins critical for commitment to apoptosis. These interactions will be examined in the absence of detergents, conditions in which the protein structures are preserved, using Bcl-2 cleavage in vivo. Direct protein-protein interactions will be examined by immunolocalization and immunoprecipitations combined with immunoblot analyses. The interactions of Bcl-2 will be also examined in cells engineered to express Bcl-2 inducibly and cells in which Bcl-2 has been targeted to specific subcellular locations. SPECIFIC AIM 3: To determine the role of Bcl-2 in regulating mitochondrial events necessary for commitment to apoptosis: These experiments will evaluate: (i) cytochrome c release and cytosolic complex activation, and (ii) the role of mitochondrial membrane potential and mitochondrial permeability pore transition in mediating signals from IR and Bcl-2. Our long-term goals are to define the critical cellular signals essential for activation of caspases leading to apoptosis. By understanding the IR-triggered signals and the critical point of cellular commitment to apoptosis we may ultimately be able to specifically enhance caspase activation and cell death to overcome radioresistance in radiotherapy.